1. Field of the Invention
This invention is directed to a device for assessing the vitality of nematodes as a measure of the efficacy of potential nematicidal and nematistatic agents.
The assay device comprises a solid support coated with a gellable polymer intimately mixed with particular chemicals that serve to stabilize the assay. Included in the device are disks impregnated with a particular chemical that is an attractant for nematodes. Included in the invention are methods for using the aforementioned device in chemotaxis assays, such assays being useful for assessing the efficacy of particular compounds as nematicidal or nematistatic agents.
2. Brief Description of the Background Art
The invasion of plant roots by parasitic nematodes leads to destruction of the plant. This is a serious problem in the agricultural industry world wide. For example, the genus Meloidoqvne can infect more than 2000 different species of plants R. S. Hussey, Advanced Treatise on Meloidogyne, vol. 1, North Carolina State Univ /Graphics, Raleigh, N.C., 1985.
It is well known that the invasion of host cells is preceded by the accumulation of plant-parasitic nematodes around plant roots. H. B. Jansson et al., J. Gen. Microbiol. 112:89 (1979). It has long been recognized that one approach to preventing invasion of plant roots by nematodes is to interrupt the mechanisms of such accumulation.
It is recognized that plant-parasitic nematodes utilize specific recognition mechanisms for finding their hosts and prey in the soil. The general consensus is that, in nematodes, the primary food-finding mechanisms are governed by chemotactic factors emanating from the host. This process, which is called chemotaxis, mediates movement toward or away from chemical gradients. In no cases known to the inventors have the chemattractants emanating from plants or microorganisms been isolated and identified. Other stimuli, such as thermal, vibratory or tactile stimuli are believed to play minor roles, if any, in food-finding behavior. N. A. Croll, et al., in The Behavior of Nematodes: Their Activity, Census, and Responses, Edward Arnold, Publisher, London, 1970; B.M. Zuckerman, et al., Ann. Rev. Phytopathol. 22:95-113 (1984).
There is no direct evidence on the manner in which reception of chemotactic signals leads to an oriented response, but a recently proposed hypothesis on nematode chemoreception speculates that carbohydrates exuded by chemosensory sensillae bind to the cuticle glycocalyx and thereafter interact with the chemotactic factors, and thereby play a role in chemoreception. B. M. Zuckerman, 1984 supra. Another hypothesis speculates that, following binding of platn chematractants to receptors in the sensory process, there results an influx of both sodium and calcium ions via ion pumps, thereby causing the generation of action potentials. K. A. Wright, J. Nematol. 15:151-158 (1983).
At present parasitic nematodes are attacked by chemical agents with nematicidal or nematistatic properties, in order to prevent their invastion of plant roots. The commonly used compounds are toxic, not only to plant parasitic nematodes, but to animals (including man) as well.
For example, one of the o-(methylcarbamoyl) oximes, aldicarb (Temik.RTM.10G Union Carbide), is registered for use on several crops for the control of insect and nematode pests. The nematicidal properties of aldicarb against the root-knot nematode M. incognita were first described by Spurr, et al., (Plant Dis. Rep. 50:424-5 (1966)).
The nematicides carbofuran (C. P. DiSanzo, J. Nematol 5:22-7 (1973) and carbaryl (D. W. Fenwick, Tropic Agric. Trinidad 25:125-16 (1968)) appear to interfere with the orientation that is necessary for survival of plant parasitic nematodes.
The possibility of intervening in the detection by nematodes of chemotactic signals produced by their hosts offers attractive options for novel and non-environmentally hazardous control of plant parasitic nematodes. The development of such methods of control, as well as the discovery of new nematicidal and nematistatic agents that are more selectively toxic for nematodes, requires rapid screening procedures to allow evaluation of large numbers of potential nematicidal or nematistatic compounds. Field testing procedures are incompatible with such mass screening programs since they are cumbersome, expensive and potentially hazardous. Thus, there is a need for a rapid, reproducible, inexpensive and safe in vitro assay method for screening the effects of a large variety of substances on nematode vitality.
Many researchers have utilized nematode movement as a criterion for in vitro screening of nematicidal and nematistatic compounds. Nematodes exposed to a toxicant are classified as either motile or non-motile. Lack of motility often is equated with inability to infect or with death, and the potency of a candidate compound is thereupon evaluated. A. Hough et al., J. Nematol. 7:221-229 (1975).
One complication of such assays is that under laboratory conditions, nematodes exposed to low concentrations of a toxicant may recover and become active when the toxicant is removed. For example, the in vitro evaluation of various nonfumigant nematicides with cholinesterase-inhibiting properties can be misleading when their action is assessed by nematode motility tests which involve removal of the toxicant prior to assay. Furthermore, cholinesterase-inhibiting toxicants exhibit reversible reaction with the substrate, and treated nematodes often recover fully after the toxicant is removed. C. C. Yu et al., J. Agric. Food Chem. 25:537-540 (1972).
An additional complication to in vitro assays is that many organophosphates and carbamates will kill at very high doses. It is believed that the mode of action of sublethal doses of these compounds is nematistatic, and it is known that intoxication with carbamate nematicides is reversible at sublethal dosages. A. Hough, 1975, supra.
Thus, there is need for a reproducible in vitro assay method that is sufficiently rapid so as to obviate the complications of reversibility of the toxic effects of some nematicidal and nematistatic agents.
Assays employed in studies of chemotactic agents are potentially suitable for the rapid in vitro assessment of the potential of candidate nematicides and nematistatic agents. Nematode chemotaxis assay methods and devices are disclosed by S. W. Ward (Proc. Nat. Acad. Sci.70:817-21 (1973)), J. Balan et al., (Nematologica 22:306-11 (1976)), J.-C. Prot (Rev. Nematol. 1:21-6, 135-142 (1978); (Rev. Nematol. 2:11-21 (1979)), H.-B. Jansson, et al.,(J. Gen. Microbiol. 112:89-93 (1979)) and J. Schmidt, et al. (Environ. Entomol. 7:605-7(1978)), and are reviewed by D. B. Dusenbery (J. Nematol 15:168-173 (1983)). All devices, except that of Prot supra, comprise Petri dishes coated with a layer of agar, agarose or Sephadex, the material of the coating being suspended initially in either sterile water or water-based HEPES buffer containing a detergent. Generally, the chemattractant in a suitable solvent is placed upon the surface of the coating at a prescribed location. The point of application of the chemattractant is usually covered with a disc of paper or agar. During a diffusion period of from 24 to 48 hours, gradients of the chemattractant are formed in the coating. A water or buffer control is applied to another site on the coating. Thereafter, a suspension of nematodes is placed at an appropriate site on the plate, and movement of nematodes toward the attractant is followed.
The device of Prot supra comprises a U-tube containing in both arms a continuous column of agar. The method comprises the addition of a chemattractant to one arm of the tube, and the formation of a gradient during a 48-hour diffusion period; water is added to the other arm as a control. Thereafter, nematodes are introduced into the bottom center of the agar column, and the number of nematodes moving to the chemattractant is determined and compared to the number moving to the control.
In spite of these methods and devices, it would still be useful to provide improved devices, and methods for using same, that aid in rapidly assessing the efficacy of large numbers of potential nematicidal and nematistatic agents in coated-plate motility assay systems that are based upon the principle of chemotaxis. Furthermore, the identification and use of specific nematode chemattractants in such assay systems may permit the discovery or development of new compounds which offer greater selectivity for plant parasitic nematodes and at the same time less toxicity for beneficial insects, as well as for animals and man.